DESCRIPTION ( Investigator's abstract): The goal of this research is to develop a gene-based therapy for metabolic deficiency disorders using cutaneous cells as a vehicle for systemic delivery. New genetic material would be introduced into epidermal keratinocytes and dermal fibroblasts to create a cutaneous source of secreted protein. For example, the hypercholesterolemia and atherosclerosis of type III hyperlipoproteinemia may be ameliorated by creation of a cutaneous source of apolipoprotein E3 (apoE). The question posed here is, can sufficient protein be produced and delivered to the circulation to ameliorate such a disorder? To date, the Principal Investigator has made substantial progress in development of methods for ex vivo and in vivo gene transfer, using retrovirus vectors in analysis of the secretory capacity of fibroblasts and keratinocytes, and in creating culture and animal models for quantitative analysis and efficacy testing. The Principal Investigator has succeeded in transducing keratinocyte stem cells through ex vivo and in vivo approaches and has demonstrated long term (10 month) reporter gene expression in animal models. In this revised application three specific aims are proposed. In Aim 1, studies are proposed to optimize systemic replacement therapy through ex vivo gene transfer. First a system that allows repeated monitoring of secreted protein in the same animal will be developed and then that system will be used to develop predictive in vitro tests, to examine dermal versus epidermal delivery, and to gauge if lymphatic uptake is rate limiting. As proof of principal, two animal models will be used, one involving apoE-deficient mice and the other involving diabetic rats. In Aim 2, the methods for in vivo transduction of mouse skin will be optimized. Specifically the use of lentivirus vectors to transduce slowly cycling stem cells in skin and application of a topical selective agent to augment the number of transduced cells in the tissue will be used. To control transgene expression in vivo, Aim 3 will involve construction of two retroviral vectors, one to permit transgene induction with topically applied inducers and the other to evade potential modulating effects of host cytokines. These studies probe the interface between cutaneous biology and corrective gene transfer and will provide a foundation for future clinical cutaneous gene therapy trials.